Clear cell renal carcinoma (ccRCC) is the 8th leading cause of cancer death in the United States. While the majority of localized RCC is cured with surgery, ~30% of patients progress to distant metastases where survival drops to <2 years and for which no curative therapies exist. Over the past five years exciting new findings revealed that genes regulating the epigenome, including SETD2, BAP1, and PBRM1, are mutated in >50% of ccRCC cases. The mechanism by which this class of mutations initiates and drives tumorigenesis, however, remains completely unknown. The epigenome is profoundly disrupted in cancer. One of the best characterized epigenetic marks is DNA methylation (5mC). 5mC is a potent transcriptional repressive signal when present in promoters and enhancers, but paradoxically is associated positively with transcription in gene bodies. During cancer progression, promoters and enhancers of growth regulatory genes are targeted for hypermethylation-mediated silencing while other regions of the genome lose 5mC, such as gene bodies. 5mC mediated by DNA methyltransferases and histone H3 lysine 36 trimethylation (H3K36me3) mediated by SETD2 co-localize in gene bodies, their levels increase with transcription, and DNMT3B binds to H3K36me3 providing a mechanistic link. Our preliminary and published data reveal a novel crosstalk between 5mC and H3K36me3 in ccRCC, in which SETD2 inactivation results in loss of 5mC targeting to gene bodies and dramatic genome-wide DNA hypermethylation, demonstrating that SETD2 mutation represents a new hypermethylator driver gene akin to IDH1/IDH2 mutation. Our long term goal is to use SETD2 as a paradigm for understanding how epigenetic-regulator mutations drive tumorigenesis and how these mutations can be targeted. Our specific hypothesis for this application is that SETD2 mutations drive ccRCC in part by causing global DNA hypermethylation that promotes a more aggressive, metastatic gene expression program. In addition, we hypothesize that this class of ccRCCs is susceptible to DNA hypomethylating agents. We will address this hypothesis with three aims. In aim 1 we will characterize interactions between 5mC/DNMTs and H3K36me3 to define the mechanism by which SETD2 mutation drives aberrant 5mC targeting and gene expression. In aim 2 we interrogate how SETD2 mutation-mediated deregulation of 5mC and transcriptional patterns impact cell growth, metastasis, and susceptibility to epigenome-targeting agents. Finally in aim 3 we define 5mC signatures linked to EMT, and validate and characterize key 5mC deregulation events through which loss of H3K36me3 activity drives a metastatic gene expression program. Our studies will shed new light on how the genome and epigenome interact and yield a detailed picture of how mutation of an epigenetic regulator gene drives tumorigenesis. This is expected to positively affect human health by allowing for a more complete understanding of the molecular etiology underlying cancers with epigenetic regulator mutations and drive discovery of new therapies that specifically target the pathways they deregulate.